CN111426658B - Method for detecting thrombin by utilizing quantum dot sensitized up-conversion nano material - Google Patents
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Abstract
The invention discloses a method for detecting coagulation by utilizing quantum dot sensitized up-conversion nano materialA method of enzyme comprising the steps of: (1) Preparing aptamer TBA1 modified up-conversion nano particles (UCNPs-TBA 1); (2) Preparation of aptamer TBA2 modified Ag 2 Se quantum dot (Ag) 2 Se QDs-TBA 2); (3) drawing a standard curve of thrombin detection; (4) detecting the thrombin concentration in the sample to be tested. The invention solves the problem of low signal-to-back ratio detected by using the FRET sensor, realizes high-sensitivity detection of thrombin in a sample to be detected, and has the detection limit of 0.091nM.
Description
Technical Field
The invention belongs to the field of biosensing and analysis, and particularly relates to a method for realizing fluorescence detection of thrombin by utilizing quantum dot sensitized up-conversion nano materials.
Background
The rare earth ion doped up-conversion nanoparticle can continuously absorb two or more low energy photons, emitting one high energy photon. The property of long wave excitation and short wave emission can effectively avoid the interference of autofluorescence and scattered light from a biological sample, so that the up-conversion nano particles are widely applied to the field of biological analysis and detection. In addition, because of the feature of avoiding simultaneous excitation with other substances, the up-conversion nanoparticles are often used in combination with Fluorescence Resonance Energy Transfer (FRET) technology as an energy donor for FRET systems, while nanomaterials with larger molar absorptivity such as nanogold, manganese dioxide, and carbon nanomaterials are typically used as an energy acceptor for FRET systems. In the detection process, firstly, an energy donor-acceptor is assembled to construct a FRET system, and fluorescence of the up-conversion nano particles is quenched by the acceptor; when the target appears, the fluorescence of the up-conversion nano particles is recovered by changing the absorption spectrum of the receptor or the distance between the donor and the receptor, so that the detection of the target is realized. It follows that this response mode based on "quench-rise" is limited in detection sensitivity by quench efficiency and rise efficiency.
Since the particle size of the up-conversion nanoparticle is usually tens of nanometers, the luminescent ions are doped in the matrix lattice, and the structural characteristic makes the spatial distance between the luminescent ions serving as energy donors and external energy acceptors be far, and the spatial distance exceeds the effective distance range where energy transfer occurs, so that the energy transfer efficiency is low, and the fluorescence quenching degree is limited. While carbon nanomaterials generally have higher quenching efficiency, the fluorescence recovery process is limited due to their strong non-specific adsorption of the upconverting nanoparticles. Both low quenching efficiency and low lifting efficiency severely limit the signal-to-back ratio and sensitivity of the detection. In view of the above, there is still a need in the art to find new detection methods to improve the signal-to-back ratio and realize high-sensitivity detection of tumor markers.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for detecting thrombin by utilizing quantum dot sensitized up-conversion nano particles, quantitatively detects thrombin in diluted serum, solves the problem of low signal-to-back ratio detected by utilizing a FRET sensor, and realizes high-sensitivity detection of thrombin in serum, wherein the detection limit can reach 0.091nM.
The technical scheme provided by the invention for solving the technical problems is as follows:
a method for detecting thrombin by using quantum dot sensitized up-conversion nano-particles, comprising the following steps:
(1) Preparation of aptamer TBA1 modified up-conversion nanoparticles: preparing surface oleic acid coated up-conversion nano particles by taking rare earth oleate as a precursor through a coprecipitation method, removing ligands to obtain up-conversion nano particles without ligand modification and with exposed surfaces, carrying out surface functionalization on the surfaces of the up-conversion nano particles by polyacrylic acid to enable the surfaces of the up-conversion nano particles to be rich in carboxyl groups, and reacting the up-conversion nano particles with an aptamer TBA1 with one end modified with amino groups after the surface functionalization to obtain TBA1 modified up-conversion nano particles;
(2) Preparation of aptamer TBA2 modified Ag 2 Se quantum dots: carboxyl modified Ag 2 Coupling reaction is carried out on the Se quantum dots and an aptamer TBA2 with one end modified with amino to obtain the Ag modified by TBA2 2 Se quantum dots;
(3) Drawing a standard curve of thrombin detection: up-conversion nano particles obtained in the step (1) and the step #2) The Ag obtained 2 Se quantum dots are added into a buffer solution, thrombin with different amounts is added into the buffer solution for incubation, the incubated solution is placed into a cuvette and excited by a 980nm laser source to obtain fluorescence intensity, and when the concentration of the thrombin is set to be 0, the obtained blank sample intensity is recorded as F 0 At a fluorescence ratio F/F 0 Drawing a standard curve by taking the concentration of thrombin in the buffer solution as an abscissa;
(4) Diluting a sample to be detected with a buffer solution, measuring the fluorescence intensity under the same condition as in the step (3), and further obtaining the thrombin concentration in the sample to be detected according to the standard curve obtained in the step (3).
According to the scheme, the up-conversion nano particles have absorption spectrum and Ag 2 The emission spectra of Se quantum dots overlap, and Ag 2 Se quantum dots have absorption at 980 nm.
According to the scheme, the up-conversion fluorescent nanoparticle consists of NaYF 4 Yb and Er, the morphology is spherical, the grain diameter is 20-28nm, and the crystal phase is hexagonal; the Ag is 2 The Se quantum dot has the particle size of about 3-4nm and the morphology of spherical.
According to the scheme, the 5' ends of the single-stranded nucleic acid TBA1 and the single-stranded nucleic acid TBA2 are respectively modified with amino groups, and the sequences of the amino groups are as follows: 5' -NH 2 TTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3 'and 5' -NH 2 -TTTTTGGTTGGTGTGGTTGG-3’。
According to the scheme, the Ag 2 The ratio of Se quantum dots to TBA2 in the coupling reaction is 5nmol: (0.5-3) nmol.
According to the scheme, the buffer solution is HEPES buffer solution, the concentration is 10mM, and the pH is 7.2. The buffer solution can also be replaced by serum diluted by the buffer solution, and the dilution factor of the serum is 20-100 times.
According to the above scheme, in step (3), thrombin is added to the buffer solution in an amount of 0 to 125nM based on the concentration of thrombin in the buffer solution.
According to the scheme, in the step (3), the concentration of the up-conversion nano particles obtained in the step (1) in a buffer solution is 0.04-0.06mg/mL; ag obtained in step (2) 2 The concentration of Se QDs-TBA2 in the buffer solution is 0.08-0.4 mu M.
According to the scheme, the incubation time is 2-5h, and the incubation temperature is 37 ℃.
The principle of the invention is as follows: the sandwich type nucleic acid aptamer sensor for detecting thrombin is constructed by utilizing the fact that thrombin contains two different DNA aptamer binding sites and by the specific recognition effect of an aptamer. Two aptamers (TBA 1 and TBA 2) of thrombin are respectively modified on the surfaces of up-conversion nano particles and quantum dots, when thrombin exists in a system, the TBA1 and TBA2 specifically recognize the thrombin to form an aptamer secondary structure, the distance between the quantum dots and the up-conversion nano particles is shortened, the quantum dots transfer the excited state energy of the quantum dots to the up-conversion nano particles, the luminescence of the up-conversion nano particles is enhanced, and the enhanced degree of the up-conversion luminescence is positively correlated with the concentration of the thrombin, so that quantitative detection of the thrombin is realized.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention can directly detect in serum samples by utilizing the characteristic of near infrared excitation and visible emission of the up-conversion fluorescent nano material, does not need a pre-treatment step, has simple operation and is more suitable for practical application.
2. According to the invention, the relative concentration of the quantum dots and the aptamer is optimized, so that the fluorescence enhancement multiple of the system can be effectively improved;
3. the invention utilizes the advantage of large absorption coefficient of quantum dots, can effectively enhance the luminescence of the up-conversion nano material, improves the detection signal-to-back ratio, solves the problem of low signal-to-back ratio detected by using a FRET sensor, realizes the high-sensitivity detection of thrombin in serum, and has the detection limit of 0.091nM.
Drawings
FIG. 1 is a schematic diagram of detection of thrombin using quantum dot sensitized up-conversion nanomaterials.
FIG. 2 is a graph showing the fold increase in fluorescence intensity with thrombin concentration over time.
FIG. 3 is a graph showing the relationship between thrombin concentration and fold increase in fluorescence after different amounts of TBA2 label quantum dots; wherein the figure is a graphThe ratio in the examples is Ag 2 Molar ratio of Se QDs and TBA 2.
FIG. 4 is a graph showing the relationship between thrombin concentration and fold increase in fluorescence after reaction of different amounts of TBA 2-QDs.
FIG. 5 is a graph showing the relationship between thrombin concentration and fold increase in fluorescence in serum at various dilutions.
Detailed Description
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments. In the following examples, the up-conversion nanomaterial and modification thereof were performed as follows:
1. oil phase NaYF 4 Synthesis of Yb and Er: 6.4mL of oleic acid, 10.4mL of 1-octadecene and 0.8mmol Ln (ole) 3 (ln=y: yb: er=78:20:2) was put into a three-necked flask, and after heating to 50 ℃, 3.2mmol of NH was added 4 F and 2mmol of NaOH in 10mL of methanol, and reacting at 50 ℃ for 30min; and then raising the temperature to 100 ℃ under the protection of argon, pumping methanol gas in a three-port bottle by using a vacuum pump, raising the temperature to 290 ℃, reacting for 90min at the temperature, naturally cooling to room temperature, adding a proper amount of ethanol, centrifugally separating and collecting precipitate, washing twice by using a mixed solution of cyclohexane and ethanol with the volume ratio of 1:2, and finally dispersing the precipitate in cyclohexane. The shape of the up-conversion fluorescent nanoparticle is spherical, and the particle size is about 24nm, namely the surface oleic acid coated up-conversion nanoparticle.
Removing the ligand: taking 50mg of the synthesized surface oleic acid coated up-conversion nano particles, adding excessive ethanol, centrifuging, collecting the precipitate, adding the precipitate and 30mL of ethanol into a single-neck flask, adjusting the pH to be 1, carrying out ultrasonic treatment for 3 hours, centrifuging after ultrasonic treatment to obtain the precipitate, centrifuging and cleaning the precipitate once by using the ethanol with the pH of being 4, centrifuging and cleaning the precipitate three times by using the ethanol and ultrapure water respectively, and finally dispersing the precipitate into 20mL of ultrapure water. The process is a ligand removal process, and the up-conversion nano particles (UCNPs) without ligand modification and with exposed surfaces are obtained.
2. Modification of up-conversion nanoparticles with polyacrylic acid:
200mg of polyacrylic acid, 233mg of sodium bicarbonate and 20mL of UCNPs with 2.5mg/mL of exposed surfaces are added into a single-neck flask, the mixture is vigorously stirred for 12 hours at room temperature, the precipitate is centrifugally collected and centrifugally washed for three times by using ultrapure water, and polyacrylic acid modified up-conversion nano particles are obtained and are marked as PAA-UCNPs, and carboxyl groups are modified on the surfaces.
3. Carboxyl modified Ag 2 Se quantum dot preparation: first, 2.5. Mu.L (0.01 mmol) of TMS was stirred under inert gas 2 Se and 80mg LiN (SiMe 3 ) 2 The selenium precursor was prepared by dissolving in a mixture of 0.5mLTOP and 1mL ODE. Stirring 16.7mg (0.1 mmol) AgAc, 130 μL (0.75 mmol) 1-octanol and 5mL ODE under argon protection for one hour, slowly heating to 160deg.C, rapidly injecting selenium precursor, reacting at 130deg.C for 30min, cooling to room temperature, washing with ethanol, dispersing in nonpolar solvent to obtain the final product containing oily Ag 2 Solution of Se quantum dots.
Adding octylamine modified polyacrylate dissolved in chloroform dropwise to oily Ag 2 Fully mixing the Se quantum dots in chloroform solution, and drying the solvent at room temperature by rotary evaporation; the solid after spin-evaporation was dispersed in borate buffer (pH 12.0,50X 10) -3 M) purification by filtration with a polypropylene column packed with Superdex 200prep grade to give carboxyl-modified Ag 2 Se quantum dots.
The obtained carboxyl modified Ag 2 After EDC and NHS activate carboxyl, se quantum dot can be coupled with an aptamer TBA2 with one end modified with amino.
4. Coupling of TBA1 and PAA-UCNPs: 1mg of PAA-UCNPs was added to 1mL of a buffer solution of 2- (N-morpholinoethanesulfonic acid MES (10 mM, pH=5.5), sonicated for 5min, then added with 0.5mg of EDC. HCl and 1mg of Sulfo-NHS, after shaking for 40min at room temperature, the precipitate was centrifuged and washed twice with ultra pure water, and the precipitate was dispersed in 1mL of a buffer solution of HEPES (10 mM, pH=7.2), 1nmol of TBA1 was added, and shaking was performed overnight to give a coupled product. The coupled product was still collected by centrifugation and washed twice with ultra pure water and finally dispersed in 1mL Tris buffer (10 mm, ph=7.4), designated TBA1-UCNPs, and stored at 4 ℃ for further use.
Example 1
A method for detecting thrombin by using quantum dot sensitized up-conversion nano material, comprising the following steps:
(1) Preparing a TBA1-UCNPs solution as described above;
(2) TBA2 and Ag 2 Coupling of Se QDs: 5nmol of Ag 2 Se QDs were added to 1mL of PBS buffer (10 mM, pH=6.8), and after 5min of sonication, 10mg of EDC. HCl and 5mg of Sulfo-NHS were added thereto, and the mixture was shaken at room temperature for 30min. Activated quantum dots were collected by centrifugation and washed twice with PBS buffer (10 mm, ph=7.2) and then dispersed in 1mL of PBS buffer (10 mm, ph=7.2) containing 2nmol TBA2. Then, the reaction mixture was incubated at room temperature for 4 hours with shaking to give a coupled product. Finally, the coupled product was washed three times with PBS buffer (10 mm, ph=7.2) and dispersed in 1mL Tris buffer (10 mm, ph=7.4), designated TBA2-QDs, and stored at 4 ℃ for later use.
(3) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH 7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, and thrombin was added in an amount of 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025), and incubated at 37℃for 2 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(4) Drawing a thrombin detection standard curve: to 0.2mLHEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025) of thrombin was added, and incubated at 37℃for 3 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(5) Mapping thrombin detectionStandard curve: to 0.2mLHEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025) of thrombin was added, and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(6) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, and thrombin was added in an amount of 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025), and incubated at 37℃for 5 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
From the standard curves drawn for the different incubation times above, it can be seen that: the length of the incubation period will affect the fold increase of the up-conversion fluorescence, as shown in fig. 2, as the incubation period is extended to 4h, the fold increase reaches a maximum value, and the extension of the incubation period does not have a significant fluorescence increase, so that it is preferable to determine that the incubation period is 4h.
Example 2
A method for detecting thrombin by using quantum dot sensitized up-conversion nano material, comprising the following steps:
(1) Preparing a TBA1-UCNPs solution as described above;
(2) TBA2 and Ag 2 Coupling of Se QDs: 5nmol of Ag 2 Se QDs were added to 1mL of PBS buffer (10 mM, pH=6.8), and after 5min of sonication, 10mg of EDC. HCl and 5mg of Sulfo-NHS were added thereto, and the mixture was shaken at room temperature for 30min. Activated quantum dots were collected by centrifugation and washed twice with PBS buffer (10 mM, pH=7.2) and then dispersed in a buffer containing 0.5-3nmol TBA2 (respectively0.5,1,2,3) in 1mL of PBS buffer (10 mm, ph=7.2). Then, incubation was performed for 4h with shaking at room temperature, and finally, the resulting coupled products were washed three times with PBS buffer (10 mM, pH=7.2) and dispersed in 1mL Tris buffer (10 mM, pH=7.4), designated as TBA2-QDs-0.5, TBA2-QDs-1, TBA2-QDs-2, TBA2-QDs-3, respectively, and stored at 4℃for use.
(3) Drawing a thrombin detection standard curve: to 0.2mLHEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.5.064 nmol of TBA2-QDs-0.5 obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which had a value of 0,0.002,0.01,0.02,0.025) of thrombin was added, and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(4) Drawing a thrombin detection standard curve: to 0.2mLHEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA 2-QDs-1.064 nmol obtained in step (2) were added, respectively, and thrombin was added in an amount of 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025), and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(5) Drawing a thrombin detection standard curve: to 0.2mLHEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs-2 obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which had a value of 0,0.002,0.01,0.02,0.025) of thrombin was added, and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(6) Drawing a thrombin detection standard curve: to 0.2mLHEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs-3 obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which had a value of 0,0.002,0.01,0.02,0.025) of thrombin was added, and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
Ag modified by the above different amounts of aptamer TBA2 2 As shown in fig. 3, the comparison shows that: with 5nmol of Ag 2 When Se QDs and 2nmol TBA2 are labeled, the maximum enhancement of up-conversion fluorescence can be obtained, and then the amount of TBA2 is increased, so that the fluorescence enhancement is not further improved, and the amount of TBA2 in the labeling process is determined to be 2nmol.
Example 3
A method for detecting thrombin by using quantum dot sensitized up-conversion nano material, comprising the following steps:
(1) Preparing a TBA1-UCNPs solution as described above;
(2) TBA2 and Ag 2 Coupling of Se QDs: 5nmol of Ag 2 Se QDs were added to 1mL of PBS buffer (10 mM, pH=6.8), and after 5min of sonication, 10mg of EDC. HCl and 5mg of Sulfo-NHS were added thereto, and the mixture was shaken at room temperature for 30min. Activated quantum dots were collected by centrifugation and washed twice with PBS buffer (10 mm, ph=7.2) and then dispersed in 1mL of PBS buffer (10 mm, ph=7.2) containing 2nmol TBA2. Then, incubation was performed for 4h with shaking at room temperature, and finally, the coupled product was washed three times with PBS buffer (10 mm, ph=7.2) and dispersed in 1mL Tris buffer (10 mm, ph=7.4) and stored at 4 ℃ for later use.
(3) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.016nmol of TBA2-QDs obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0)025) thrombin, incubated at 37℃for 4h. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(4) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.032nmol of TBA2-QDs obtained in step (2) were added, and thrombin was added in an amount of 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025), and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(5) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.048nmol of TBA2-QDs obtained in step (2) were added, respectively, 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025) of thrombin was added, and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(6) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, and thrombin was added in an amount of 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025), and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(7) Drawing a thrombin detection standard curve: to 0.2mL of HEPES buffer (10 mM, pH=7.2), 0.01mg of TBA1-UCNPs obtained in step (1) and 0.08nmol of TBA2-QDs obtained in step (2) were added, and thrombin was added in an amount of 0 to 0.025nmol (each of which was 0,0.002,0.01,0.02,0.025), and incubated at 37℃for 4 hours. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
By adjusting the amount of TBA2-QDs added, as shown in FIG. 4, the concentration of TBA2-QDs can be increased by a multiple of fluorescence enhancement, but when the concentration of TBA2-QDs in the buffer solution is more than 0.32. Mu.M (i.e., the amount of TBA2-QDs added during the process of drawing a standard curve is 0.064 nmol), the multiple of fluorescence enhancement is decreased instead, because excessive QDs-TBA2 causes an internal filtration effect inside the system, and the up-converted luminescence is quenched instead, so that the optimal concentration of QD-TBA2 in the buffer solution can be obtained to be 0.32. Mu.M.
Example 4
A method for detecting thrombin by using quantum dot sensitized up-conversion nano material, comprising the following steps:
(1) Preparing a TBA1-UCNPs solution as described above;
(2) TBA2 and Ag 2 Coupling of Se QDs: 5nmol of Ag 2 Se QDs were added to 1mL of PBS buffer (10 mM, pH=6.8), and after 5min of sonication, 10mg of EDC. HCl and 5mg of Sulfo-NHS were added thereto, and the mixture was shaken at room temperature for 30min. Activated quantum dots were collected by centrifugation and washed twice with PBS buffer (10 mm, ph=7.2) and then dispersed in 1mL of PBS buffer (10 mm, ph=7.2) containing 2nmol TBA2. Then, incubation was performed for 4h with shaking at room temperature, and finally, the coupled product was washed three times with PBS buffer (10 mm, ph=7.2) and dispersed in 1mL Tris buffer (10 mm, ph=7.4) and stored at 4 ℃ for later use.
(3) Serum dilution: fresh serum was obtained from the hospital and diluted 20-fold, 50-fold, 75-fold, 100-fold with HEPES buffer (10 mM, pH=7.2), respectively, and designated HEPES-server 20, HEPES-server 50, HEPES-server 75, HEPES-server 100, and stored at 4℃for further use.
(4) Drawing a thrombin detection standard curve: to 0.2mL of HEPES-server 20, 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, and 0 to 0.025nmol (0, 2X 10, respectively) was added -5 ，2×10 -4 0.001,0.003,0.008,0.015,0.02,0.025) thrombin, incubated for 4h at 37 ℃. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(5) Drawing a thrombin detection standard curve: to 0.2mL HEPES-server 50, 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, respectively, 0-0.025nmol (0, 2X 10, respectively) -5 ，2×10 -4 0.001,0.003,0.008,0.015,0.02,0.025) thrombin, incubated for 4h at 37 ℃. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(6) Drawing a thrombin detection standard curve: to 0.2mL of HEPES-server 75, 0.01mg of TBA1-UCNPs obtained in step (1) and 0.064nmol of TBA2-QDs obtained in step (2) were added, and 0 to 0.025nmol (0, 2X 10, respectively) was added, respectively -5 ，2×10 -4 0.001,0.003,0.008,0.015,0.02,0.025) thrombin, incubated for 4h at 37 ℃. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
(7) Drawing a thrombin detection standard curve: adding the TBA1-UCNPs 0.0 obtained in the step (1) to 0.2mL HEPES-server 1001mg and 0.064nmol of TBA2-QDs obtained in the step (2) are added respectively with 0-0.025nmol (the values are 0,2×10 respectively) -5 ，2×10 -4 0.001,0.003,0.008,0.015,0.02,0.025) thrombin, incubated for 4h at 37 ℃. Subsequently, up-converted fluorescence was measured at 546nm under 980nm continuous wave laser excitation. When the thrombin concentration was set to 0, the blank strength was designated as F 0 At a fluorescence ratio F/F 0 The thrombin standard solution concentration is plotted on the ordinate as the abscissa.
By comparing the above dilutions of serum with HEPES buffer at different fold ratios, as shown in fig. 5, it can be seen that: the fold increase in upconversion fluorescence increases with increasing serum dilution due to the presence of a large number of interferents in human serum that prevent aggregation and energy transfer between QDs and UCNPs, but a linear thrombin detection range of 0.1nM to 75nM with a limit of detection of 0.091nM is obtained in 100-fold diluted serum.
Example 5
TABLE 1
As shown in Table 1, according to the experimental conditions optimized in examples 1-4 and the standard curve for thrombin detection (i.e., the standard curve established in step (7) of example 4), in the standard recovery experiment of 100-fold diluted serum samples, the addition amount of thrombin was 1.5, 5, 10 and 30nM, respectively, resulting in a standard recovery rate of 91.93% -102.8% and a relative standard deviation of 0.749% -6.97%, which indicates the feasibility of the method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles in clinical analysis.
Example 6
TABLE 2
As shown in Table 2, the university of Wuhan will be followed from the middle southHospital supplied plasma samples are pre-treated in the existing way to convert prothrombin to prothrombin. Since prothrombin is present in plasma as its precursor, 0.03M CaCl is added to this plasma sample 2 The prothrombin was converted to prothrombin, left to stand for 2 hours for activation, and then plasma containing thrombin was diluted 100-fold with HEPES buffer (10 mM, pH 7.2).
Thrombin in the plasma samples was then tested according to the experimental conditions optimized for examples 1-4 and the standard curve for thrombin detection (i.e. the standard curve established in step (7) of example 4). By detecting thrombin levels in 4 human plasma samples, the method of the invention yields detection results within the concentration range of thrombin in normal plasma. Compared with the test results of commercial ELISA kits, since the t-test value based on the t-test statistical analysis is smaller than the t-critical value (t crit [0.05，4]=2.78), the method for detecting thrombin by using quantum dot sensitized up-conversion nanoparticles according to the present invention is reliable in accuracy of detecting thrombin in an actual sample.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and changes can be made by those skilled in the art without departing from the inventive concept and remain within the scope of the invention.
< 110 > North university of lake
< 120 > a method for detecting thrombin by quantum dot sensitization up-conversion nano material
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<213>artificial sequence
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tttttagtcc gtggtagggc aggttggggt gact 34
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Claims (7)
1. A method for detecting thrombin by using quantum dot sensitized up-conversion nano-particles, which is characterized by comprising the following steps:
(1) Preparing aptamer TBA1 modified up-conversion nano particles;
(2) Preparation of aptamer TBA2 modified Ag 2 Se quantum dots: ag with 2 Reacting Se quantum dots with an aptamer TBA2 with one end modified with amino to obtain TBA2 modified Ag 2 Se quantum dots;
(3) Drawing a standard curve of thrombin detection: up-converting the nano particles obtained in the step (1) and Ag obtained in the step (2) 2 Adding Se quantum dots into a buffer solution, adding thrombin with different amounts into the buffer solution for incubation, exciting the incubated solution by using a laser light source to obtain fluorescence intensity, and recording the obtained blank sample intensity as F when the concentration of the thrombin is set to be 0 0 At a fluorescence ratio F/F 0 Drawing a standard curve by taking the concentration of thrombin in the buffer solution as an abscissa;
(4) Diluting a sample to be detected with a buffer solution, measuring the fluorescence intensity under the same condition as that of the step (3), and further obtaining the thrombin concentration in the sample to be detected according to the standard curve obtained in the step (3);
the aptamers TBA1 and TBA2 are respectively aptamers which specifically recognize two different DNA aptamer binding sites of thrombin; aptamer TBA1 and TBA2 modified up-conversion nanoparticle and Ag, respectively 2 When thrombin exists in a system on the surface of Se quantum dots, TBA1 and TBA2 specifically recognize thrombin to form an aptamer secondary structure; absorption spectrum and Ag of the up-conversion nano-particles 2 The emission spectra of Se quantum dots overlap;
the chemical formula of the up-conversion fluorescent nanoparticle is NaYF 4 Yb and Er are spherical in shape and 20-28nm in particle size; the Ag is 2 The Se quantum dot has a particle size of 3-4nm, a spherical shape and a carboxyl group on the surface.
2. The method for detecting thrombin by using quantum dot sensitized up-conversion nano particles according to claim 1, wherein the 5' ends of the single-stranded nucleic acid TBA1 and the single-stranded nucleic acid TBA2 are modified with amino groups, and the sequences are respectively as follows: 5' -NH 2 TTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3 'and 5' -NH 2 -TTTTTGGTTGGTGTGGTTGG-3’。
3. The method for detecting thrombin by using quantum dot sensitized up-conversion nano particles according to claim 1, wherein in the step (3), the buffer solution is HEPES buffer solution, and the pH is 7.0-7.5.
4. The method for detecting thrombin by quantum dot sensitized up-conversion nano particles according to claim 1, wherein in the step (3), the amount of thrombin added in the buffer solution is 0 to 125nM in terms of its concentration in the buffer solution.
5. The method for detecting thrombin by quantum dot sensitized up-conversion nano particles according to claim 1, wherein in the step (3), ag obtained in the step (2) 2 The addition amount of Se quantum dots is 0.08-0.4 mu M based on the concentration of Se quantum dots in the buffer solution.
6. The method for detecting thrombin by quantum dot sensitized up-conversion nanoparticle according to claim 1, wherein in the step (3), the incubation time is 2-5h and the incubation temperature is 37 ℃.
7. The method for detecting thrombin by using quantum dot sensitized up-conversion nano-particles according to claim 1, wherein the sample to be detected is a plasma sample, and the pretreatment is needed to convert prothrombin into thrombin.
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